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10.1126/science.291.5512.2364
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of April 24, 2012 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/291/5512/2364.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/291/5512/2364.full.html#related This article cites 71 articles, 28 of which can be accessed free: http://www.sciencemag.org/content/291/5512/2364.full.html#ref-list-1 This article has been cited by 836 article(s) on the ISI Web of Science This article has been cited by 100 articles hosted by HighWire Press; see: http://www.sciencemag.org/content/291/5512/2364.full.html#related-urls This article appears in the following subject collections: Cell Biology http://www.sciencemag.org/cgi/collection/cell_biol Downloaded from www.sciencemag.org on April 24, 2012
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REVIEW
the glycans have a common role in promoting protein folding, quality control, and certain sorting events. Later, Golgi enzymes prepare them for the spectrum of novel functions that the sugars display in the mature proteins (3). Here, we mainly address events in the early secretory pathway. We focus on observations that are starting to unmask the logic of the various early trimming and modification events. We also discuss glycan structure and function in light of fundamental differences between the two biosynthetic organelles, the ER and the Golgi complex.
the ER to diversification in the Golgi complex coincides with a marked change in glycan function. In the early secretory pathway,
Institute of Biochemistry, 2Institute of Microbiology, Eidgeno ssische Technische Hochschule Zu rich, Universita tstrasse 16, CH-8092 Zu rich, Switzerland. *To whom correspondence should be addressed.
Fig. 1. The N-linked core oligosaccharide. N-linked glycans are added to proteins in the ER as core oligosaccharides that have the structure shown. These are bound to the polypeptide chain through an N-glycosidic bond with the side chain of an asparagine that is part of the Asn-X-Ser/Thr consensus sequence. Terminal glucose and mannose residues are removed in the ER by glucosidases and mannosidases. The symbols for the different sugars are used in the following gures.
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Fig. 2. Biosynthesis of the N-linked core oligosaccharide. Synthesis starts on the cytosolic surface of the ER membrane by the addition of sugars, one by one, to dolichylphosphate. When two N-acetylglucosamines and ve mannoses have been added, the oligosaccharide is ipped to the lumenal side of the membrane, and seven further sugars are added from lipid precursors. After the last of the three glucoses have been added, the oligosaccharyltransferase enzyme complex catalyzes the transfer of the core oligosaccharide to the asparagine residues of nascent, growing polypeptide chains. The three glucoses are trimmed away by glucosidase I and II, and terminal mannoses by one or more different ER mannosidases. The ER also contains a glucosyltransferase that can regluco-
sylate glucose-free chains and thus establish, with glucosidase II, a deglucosylation-reglucosylation cycle. When the glycoprotein has folded (gray oval) and reached the Golgi complex, further mannose trimming occurs. The addition of a GlcNAc residue is followed by trimming of two additional mannoses. During subsequent terminal glycosylation there is addition of new terminal sugars including GlcNAc, galactose, sialic acid, and fucose. Of the original core glycan, just ve sugars remain. Only one of many possible terminal glycosylation pathways is shown; the number of branches generated is variable, as are the number and identity of sugars added. Whereas the glycoforms in the ER are homogeneous, the Golgi-generated forms are highly diverse and differ widely between species.
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Fig. 3. The calnexin-calreticulin cycle. When two of the glucoses in the N-linked core glycans have been trimmed away by glucosidases I and II, the nascent or newly synthesized glycoproteins bind to calnexin (CNX) and/or calreticulin (CRT), two homologous ER lectins specic for monoglucosylated core oligosaccharides. The protein is thereby exposed to another folding factor, ERp57, a thiol oxidoreductase that binds to both calnexin and calreticulin. If the glycoproteins have cysteines, the formation of disulde bonds is catalyzed through the formation of transient mixed disuldes with ERp57. When the remaining third glucose residue is trimmed by glucosidase II, the complexes dissociate. If the glycoprotein is not folded at this time, the oligosaccharides are reglucosylated by an ER glucosyltransferase, and the protein reassociates with the lectins. The cycle is repeated until the protein is either folded or degraded. Once correctly folded, a glycoprotein is no longer recognized by the glucosyltransferase, and because it is no longer reglucosylated, it will not bind back to calnexin and/or calreticulin. It can now leave the ER. Exit of certain glycoproteins from the ER to the Golgi complex is assisted by another membrane-bound lectin, ERGIC-
53, which binds to mannose residues. The calnexin-calreticulin cycle promotes correct folding, inhibits aggregation of folding intermediates, blocks premature oligomerization, regulates ER degradation, and provides quality control by preventing incompletely folded glycoproteins from exiting to the Golgi complex.
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Summary
Glycosylation differs from most other covalent protein modifications with respect to the size and complexity of the added group and the magnitude of the cellular machinery devoted to synthesis and modulation. Why do cells need such an elaborate and costly system? Why are so many proteins in the eukaryotic cell glycosylated? Why do the Nlinked glycans undergo so many changes during glycoprotein maturation? At a conceptual level, it is important to recognize that one of the limitations imposed by the linear nature of polypeptide chains is the lack of possibilities to generate branched structures. Addition of oligoand polysaccharides (N-linked or O-linked) provides a way to circumvent this limitation. In the case of N-linked glycans, the branches reach more than 3 nm from the protein surface as bulky, mobile carbohydrate clusters that are themselves branched. The outer parts are so far from the protein surface that they can act as essentially independent domains. One of the explicit advantages of adding these polar branches is that cells can produce and secrete proteins of greater complexity and with better efficiency than would otherwise be the case. As we have seen, many plasma membrane proteins and secretory proteins are not able to fold without added glycans. The evolution of proteins into better folders has been facilitated not only by the addition of glycans, but probably also by the generation and elimination of glycosylation sequons in the primary sequence. This can occur by single point mutation, allowing easy modulation of folding parameters through the shuffling of glycosylation sites. Because carbohydrates differ in biosynthesis and overall properties from amino acids, the joining of polypeptide and carbohydrate elements into hybrid molecules provides an opportunity to add new functionalities and specificities. Differential terminal glycosylation pathways allow, for example, fine-tuning of protein properties in a cell- and tissue-specific manner without a change in amino acid sequence. The large number of possible ways to link sugars to each other makes oligosaccharides ideal as compact and versatile recognition markers. It is not uncommon, as shown by calnexin and the M-6-P receptors, that a single sugar residue or sugar-associated group suffices as a distinctive marker for lectin binding. The more complex a eukaryotic organism, the more numerous are the ways in which it seems to make use of this opportu-
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